The invention relates to a process for the surface modification of a porous polymer monolith and products produced therefrom. In particular, the process relates to the surface modification of a polystyrenic monolith by alkylating its through-pore surfaces and to products so modified.
Liquid chromatography, as a technique for the separation of soluble, non-volatile compounds, has become over the past 30 years an indispensable tool for chemical and biochemical analyses in numerous disciplines of chemistry and life sciences. Generally, separation in liquid chromatography is achieved in a column by selective distribution of the sample molecules between a stationary phase and a mobile phase. In reversed-phase liquid chromatography, the stationary phase is usually highly hydrophobic or non-polar. Conventional reversed-phase liquid chromatography uses 5-10 xcexcm spherical silica beads that have been modified by covalent attachment of hydrocarbon chains including 4, 8, or 18 carbon atoms to provide a non-polar surface.
In addition to silica beads, PS-DVB particles are also widely used as a stationary phase support. Actually, the PS-DVB surface can be directly used as the reversed-phase chromatography stationary phase since it is highly hydrophobic. Maa and Horvxc3xa1th, J. Chromatography, 445 (1988), 71-86, disclose that PS-DVB particles are very effective for the rapid analysis of proteins in the reversed-phase mode. However, for separation and identification of smaller biomolecules like peptides, which have become more crucial to the new emerging field of proteomics, the unfunctionalized PS-DVB particles may present unacceptable poor resolution. It has been shown by Huber et al., Chromatographia, 44 (7/8) (1997), 438-448, that alkylation of PS-DVB particles to graft octadecyl chains on their surface is necessary to achieve good resolution for reversed-phase liquid chromatography of peptides.
The increasing demand for more efficient and rapid separations in many areas especially for the pharmaceutical industry has initiated research towards column consolidation and miniaturization. In recent years, column consolidation has been achieved as a result of the introduction or invention of porous polymer continuous beds or monoliths. Hjertxc3xa9n, J. Chromatography, 473 (1989), 273-275 introduces a polymer gel continuous bed prepared by in situ polymerization of an aqueous solution of acrylamide derivatives. Svec and Frxc3xa9chet disclosed in 1994 and 1995 (U.S. Pat. Nos. 5,334,310 and 5,453,185) a continuous liquid chromatographic column containing a separation medium in the form of a macroporous polymer plug. The column miniaturization has also been achieved by a porous polymer monolith prepared by free radical polymerization in situ in a fused silica capillary. The development of fritless columns with a polymer-based porous monolith rather than conventional spherical beads has become more and more important since it meets the requirement of today""s micro-scale liquid chromatography and capillary electrochromatography as described by Liao and Hjertxc3xa9n (1997 U.S. Pat. No. 5,647,979); Peters et al., Analytical Chemistry, 70 (1998), 2288-2295; Gusev et al., J. Chromatography A, 855 (1999), 273-290; and Zhang et al., J. Chromatography A, 887 (2000), 465-477.
Practicable methods for preparing a PS-DVB monolith have been published. The PS-DVB monolith has become an acceptable consolidated column packing material. However, the art lacks a method to effectively impart alkyl chains onto the through-pore surfaces of a PS-DVB monolith so as to provide an effective column packing capable of enhancing the resolution of peptides in reversed-phase liquid chromatography.
Several methods have been proposed to introduce alkyl functional groups to the polymer monolith. In one method, the alkyl groups are directly imparted from a co-monomer, e.g., alkylene dimethacrylate, rather than divinylbenzene used for the initial polymerization as claimed in U.S. Pat. No. 5,453,185 (Svec and Frxc3xa9chet). However, the introduction of a new monomer to the initial polymerization mixture may require redesign of the formulation and conditions including the selection of a new porogen. Moreover, only those alkyl chains in the polymer surfaces are needed for the separation, while the alkyl chains involved inside the bulk solid support are not necessary. It has been found that alkyl chains imparted by this process do not appreciably improve the resolution of peptides. Thus, this method for alkylating the monolith surfaces forms an ineffective stationary phase for separation of relatively smaller biomolecules like peptides.
The reaction of benzene with xe2x80x9camyl chloridexe2x80x9d in the presence of aluminum chloride to produce xe2x80x9camylbenzenexe2x80x9d was carried out by Charles Friedel and James Mason Crafts in 1877. For over a century, Friedel-Crafts alkylation chemistry has been one of the most interesting aspects of modern organic theory (R. M. Roberts and A. A. Khalaf, xe2x80x9cFriedel-Crafts Alkylation Chemistryxe2x80x9d, Mercel Dekker, Inc., New York, 1984). Major processes for the production of high-octane gasoline, synthetic rubber, plastics, and synthetic detergents are applications of Friedel-Crafts chemistry.
The Friedel-Crafts reaction has also been adopted for the surface modification of PS-DVB particles or beads to form reversed-phase column packing materials as disclosed by Huber et al., Analytical Biochemistry, 212 (1993), 351-358. According to the Huber et al. process, the solid aluminum chloride is directly added to the suspension of PS-DVB particles in an alkyl chloride (1-chlorooctadecane). No other solvent was added during the multiphase reaction. Since the reactantion is controlled by diffusion, the size of the particles which can be modified is limited.
That process is not suitable for alkylating the through-pore surfaces of a porous PS-DVB monolith. First, the solid state catalyst is difficult to introduce into the internal pores of a monolith since the solid catalyst is not very soluble in the alkylating solution. Second, the internal pores of the monolith may become clogged with precipitated solid that remains inside the monolith during the Friedel-Crafts reaction. Although there are a few liquid-state Friedel-Crafts catalysts, these are not suitable for this purpose. For example, tin(IV) chloride is a liquid and is easily filled into the monolith porous structure, but it may precipitate insoluble substances during the Friedel-Crafts reaction and it is a very weak catalyst as well. Unlike a free benzene ring which can be easily alkylated in a few minutes through the Friedel-Crafts reaction at below room temperature, a polymer-based benzene ring is much more difficult to alkylate since the Friedel-Crafts reaction is controlled by diffusion. Heating is preferred for speeding the Friedel-Crafts reaction on a PS-DVB surface.
It would be desirable, therefore, to develop a process that can be used for alkylating through-pore surfaces of a porous PS-DVB monolith as the one-piece packing material of a liquid chromatographic column. A strong catalyst solution is needed.
One aspect of the present invention relates to a process for the surface modification of a porous polystyrenic monolith. The process includes wetting the monolith internal pore (through-pore) surfaces with an organic solvent used in the uniform liquid solution. The pore surfaces of the monolith are treated by contacting the monolith pores with a uniform liquid solution containing a Friedel-Crafts catalyst, an alkyl halide, and an organic solvent so as to alkylate the internal pore surfaces. The post-reaction solution is removed from the alkylated pore surfaces by rinsing the organic solvent through the monolith. Preferably, the alkylated pore surfaces are further washed by sequentially rinsing a series of solvents through the monolith, respectively.
Another aspect of the present invention relates to a porous polystyrenic monolith having alkylated internal pore surfaces.
The present invention results in a number of advantages over the prior art. It is an advantage of the present invention to provide a simple and reliable process for alkylating internal pore surfaces of a porous polystyrenic monolith. The monolith to be alkylated may be already molded in a fused-silica capillary, a plastic tubing, a micro-channel or hole of a silicon/polymer chip, or a similar opening in a separation device. The monolith is preferably a cross-linked polystyrene copolymer having internal through-pores that a liquid can pass through.
It is a further advantage of the present invention to provide surface-alkylated porous PS-DVB monoliths as consolidated and miniaturized columns for liquid chromatographic separation, especially in the reversed-phase mode. Advantages of this invention include the improved separation and identification of peptides by using such alkylated monolithic columns in reversed-phase liquid chromatography hyphenated with electrospray ionization mass spectrometry.
Other advantages of the present invention will be apparent to those skilled in the art from the following description and the appended claims.